This invention involves an immobilized enzyme conjugate and a method of preparing such an immobilized enzyme conjugate. It is known that enzymes, which are proteinaceous in nature and which are commonly water soluble, act as biocatalysts which serve to regulate many and varied chemical reactions which occur in living organisms. The enzymes may also be isolated and used in analytical, medical and industrial applications. For example, they find use in industrial applications in the preparation of food such as cheese or bread as well as being used in the preparation of alcoholic beverages. The enzyme glucose isomerase is extensively used to convert glucose to fructose in the manufacture of high fructose corn syrup.
Since enzymes are commonly water soluble as well as being generally unstable and, therefore, subject to deactivation, they are difficult to remove for reuse from solutions in which they are utilized and they may not retain their catalytic activity over extended periods of time. These difficulties lead to an increased cost in the use of enzymes in commercial scale operations due to the necessity for frequent replacement of the enzyme. In order to reduce the high cost of enzyme replacement, various methods to immobilize or insolubilize enzymes prior to their use have been devised. This immobilization of the enzyme permits its reuse whereas it might otherwise undergo deactivation or be lost in the reaction medium in which it is used. These immobilized enzyme systems may be employed in various reactor systems, for example, in packed columns and stirred tank reactors, depending on the nature of the substrate which is being biochemically reacted.
Several general methods as well as many modifications thereof have been described by which the immobilization of enzymes can be effected.
In U.S. Pat. No. 3,796,634 there is disclosed an immobilization method which involves absorbing a polyamine onto the surface of colloidal sized particles, cross-linking the polyamine with a conventional amine-reactive cross-linking agent, e.g. glutaraldehyde, treating the resulting reaction product with NaBH.sub.4 to reduce the aldehyde groups and thereby prevent any covalent bonding between the aldehyde groups and the enzyme's amino group, and absorbing the enzyme onto the treated surface of the particle at a pH such that the colloidal absorbant bears a net electric charge opposite that of the enzyme molecules so that ionic bonding aids other non-covalent bonding forces. This patent describes the absorbant particles as ranging in size from about 50 to about 20,000 angstroms, preferably from about 100 to 200 angstroms in diameter, with the absorbant material being activated charcoal, hydroxyapatite, alumina C gamma, and betonite. This system depends on charge interactions for binding the enzyme to the treated particles. This type of bonding is less desirable than the formation of covalent linkages because ionic interactions are susceptible to the environmental conditions relative to this type of linkage such as pH, ionic strength and temperature.
Liu, et al disclose an immobilization method for lactase on granular carbon in Biotechnol. Bioeng. 17, 1695-1696, 1975 which involves absorbing p-aminophenol or 1-phenol-2-amino-4-sulfonic acid to the carbon. These absorbed compounds provide the amino groups with which glutaraldehyde reacts and in turn binds the enzyme. The amino group containing compounds mentioned are monomers which possess different chemical and physical properties than those of a polyamine such as polyethylenimine.
Another group of workers (Cho, et al, Immobilization of Enzymes on Activated Carbon: Properties of Immobilized Glucoamylase, Glucose Oxidase and Gluconolactonase, Biotechnol. Bioeng. 20, 1651-1665, 1978) have also immobilized enzymes on granular carbon by covalent attachment. In this process carbon is activated by a carbodiimide which then enables the enzyme to displace the carbodiimide and form an enzyme-carbon complex.
U.S. Pat. No. 4,141,857 (issued Feb. 27, 1979) discloses a method for enzyme immobilization which involves treating an inorganic porous support material such as gamma-alumina having pore diameters of from about 100 to about 55,000 angstroms and a surface area of about 100 to 500 m.sup.2 per gram with a solution of a water soluble polyamine and contacting the treated support material with a solution of a bifunctional monomeric material, e.g. glutaraldehyde. This treatment leaves the treated support material suitable for reaction with the enzyme so as to form covalent bonds between the enzyme and the pendant aldehyde groups. In example II of this patent there is described the preparation of an immobilized enzyme conjugate by treating porous alumina spheres sequentially with solutions of polyethylenimine, glutaraldehyde and glucoamylase.
The use of activated granular carbon has received little attention as a support for immobilized enzymes in spite of its many attractive properties and reasonable cost. Granular carbon is used industrially for purification of syrups and other food products, pharmaceutical products, organic acids and various other chemicals by continuous column percolation processes.